Abstract AMP-activated protein kinase (AMPK) is a master metabolic regulator for controlling cellular energy homeostasis. Its homolog in yeast, SNF1, is activated in response to glucose depletion and other stresses. The catalytic (α) subunit of AMPK/SNF1, Snf1 in yeast, contains a protein Ser/Thr kinase domain (KD), an auto-inhibitory domain (AID), and a region that mediates interactions with the two regulatory (β and γ) subunits. Previous studies suggested that Snf1 contains an additional segment, a regulatory sequence (RS, corresponding to residues 392–518), which may also have an important role in regulating the activity of the enzyme. The crystal structure of the heterotrimer core of Saccharomyces cerevisiae SNF1 showed interactions between a part of the RS (residues 460–498) and the γ subunit Snf4. Here we report biochemical and functional studies on the regulation of SNF1 by the RS. GST pulldown experiments demonstrate strong and direct interactions between residues 450–500 of the RS and the heterotrimer core, and single-site mutations in the RS–Snf4 interface can greatly reduce these interactions in vitro. On the other hand, functional studies appear to show only small effects of the RS–Snf4 interactions on the activity of SNF1 in vivo. This suggests that residues 450–500 may be constitutively associated with Snf4, and the remaining segments of the RS, as well as the AID, may be involved in regulating SNF1 activity.